Individualized anti-cancer antibodies

ABSTRACT

The present invention relates to a method for producing patient specific anti-cancer antibodies using a novel paradigm of screening. By segregating the anti-cancer antibodies using cancer cell cytotoxicity as an end point, the process makes possible the production of anti-cancer antibodies customized for the individual patient that can be used for therapeutic and diagnostic purposes. The invention further relates to the process by which the antibodies are made and to their methods of use. The antibodies can be made specifically for one tumor derived from a particular patient and are selected on the basis of their cancer cell cytotoxicity and simultaneous lack of toxicity for non-cancerous cells. The antibodies can be used in aid of staging and diagnosis of a cancer, and can be used to treat tumor metastases. The anti-cancer antibodies can be conjugated to red blood cells obtained from that patient and re-infused for treatment of metastases based upon the recognition that metastatic cancers are usually well vascularized and the delivery of anti-cancer antibodies by red blood cells can have the effect of concentrating the antibodies at the site of the tumor.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/415,278, filed Oct. 8, 1999, now U.S. Pat. No. 6,180,357 B1, thecontents of which are herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the production of anti-cancer antibodiescustomized for the individual patient that can be used for therapeuticand diagnostic purposes. The invention further relates to the process bywhich the antibodies are made and to their methods of use.

BACKGROUND OF THE INVENTION

Each individual who presents with cancer is unique and has a cancer thatis as different from other cancers as that person's identity. Despitethis, current therapy treats all patients with the same type of cancer,at the same stage, in the same way. At least 30% of these patients willfail the first line therapy, thus leading to further rounds of treatmentand the increased probability of treatment failure, metastases, andultimately, death. A superior approach to treatment would be thecustomization of therapy for the particular individual. The only currenttherapy which lends itself to customization is surgery. Chemotherapy andradiation treatment can not be tailored to the patient, and surgery byitself, in most cases is inadequate for producing cures.

With the advent of monoclonal antibodies, the possibility of developingmethods for customized therapy became more realistic since each antibodycan be directed to a single epitope. Furthermore, it is possible toproduce a combination of antibodies that are directed to theconstellation of epitopes that uniquely define a particular individual'stumor.

Having recognized that a significant difference between cancerous andnormal cells is that cancerous cells contain antigens that are specificto transformed cells, the scientific community has long held thatmonoclonal antibodies can be designed to specifically target transformedcells by binding specifically to these cancer antigens; thus giving riseto the belief that monoclonal antibodies can serve as “Magic Bullets” toeliminate cancer cells.

At the present time, however, the cancer patient usually has few optionsof treatment. The regimented approach to cancer therapy has producedimprovements in global survival and morbidity rates. However, to theparticular individual, these improved statistics do not necessarilycorrelate with an improvement in their personal situation.

Thus, if a methodology was put forth which enabled the practitioner totreat each tumor independently of other patients in the same cohort,this would permit the unique approach of tailoring therapy to just thatone person. Such a course of therapy would, ideally, increase the rateof cures, and produce better outcomes, thereby satisfying a long-feltneed.

Historically, the use of polyclonal antibodies has been used withlimited success in the treatment of human cancers. Lymphomas andleukemias have been treated with human plasma, but there were fewprolonged remission or responses. Furthermore, there was a lack ofreproducibility and there was no additional benefit compared tochemotherapy. Solid tumors such as breast cancers, melanomas and renalcell carcinomas have also been treated with human blood, chimpanzeeserum, human plasma and horse serum with correspondingly unpredictableand ineffective results.

There have been many clinical trials of monoclonal antibodies for solidtumors. In the 1980s there were at least four clinical trials for humanbreast cancer which produced only one responder from at least 47patients using antibodies against specific antigens or based on tissueselectivity. It was not until 1998 that there was a successful clinicaltrial using a humanized anti-her 2 antibody in combination withCisplatin. In this trial 37 patients were accessed for responses ofwhich about a quarter had a partial response rate and another half hadminor or stable disease progression.

The clinical trials investigating colorectal cancer involve antibodiesagainst both glycoprotein and glycolipid targets. Antibodies such as17-1A, which has some specificity for adenocarcinomas, had undergonePhase 2 clinical trials in over 60 patients with only one patient havinga partial response. In other trials, use of 17-1A produced only onecomplete response and two minor responses among 52 patients in protocolsusing additional cyclophosphamide. Other trials involving 17-1A yieldedresults that were similar. The use of a humanized murine monoclonalantibody initially approved for imaging also did not produce tumorregression. To date there has not been an antibody that has beeneffective for colorectal cancer. Likewise there have been equally poorresults for lung cancer, brain cancers, ovarian cancers, pancreaticcancer, prostate cancer, and stomach cancer. There has been some limitedsuccess in the use of anti-GD3 monoclonal antibody for melanoma. Thus,it can be seen that despite successful small animal studies that are aprerequisite for human clinical trials, the antibodies that have beentested have been for the most part ineffective.

PRIOR PATENTS

U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

U.S. Pat. No. 4,861,581 discloses a process comprising the steps ofobtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

U.S. Pat. No. 5,171,665 provides a novel antibody and method for itsproduction. Specifically, the patent teaches formation of a monoclonalantibody which has the property of binding strongly to a protein antigenassociated with human tumors, e.g. those of the colon and lung, whilebinding to normal cells to a much lesser degree. U.S. Pat. No. 5,484,596provides a method of cancer therapy comprising surgically removing tumortissue from a human cancer patient, treating the tumor tissue to obtaintumor cells, irradiating the tumor cells to be viable butnon-tumorigenic, and using these cells to prepare a vaccine for thepatient capable of inhibiting recurrence of the primary tumor whilesimultaneously inhibiting metastases. The patent teaches the developmentof monoclonal antibodies which are reactive with surface antigens oftumor cells. As set forth at col. 4, lines 45 et seq., the patenteesutilize autochthonous tumor cells in the development of monoclonalantibodies expressing active specific immunotherapy in human neoplasia.

U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic ofhuman carcinomas and not dependent upon the epithelial tissue of origin.

U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies which induceapoptosis in Her2 expressing cells, hybridoma cell lines producing theantibodies, methods of treating cancer using the antibodies andpharmaceutical compositions including said antibodies.

U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for theproduction of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources.

U.S. Pat. No. 5,869,268 is drawn to a method for producing a humanlymphocyte producing an antibody specific to a desired antigen, a methodfor producing a monoclonal antibody, as well as monoclonal antibodiesproduced by the method. The patent is particularly drawn to theproduction of an anti-HD human monoclonal antibody useful for thediagnosis and treatment of cancers.

U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,antibody conjugates and single chain immunotoxins reactive with humancarcinoma cells. The mechanism by which these antibodies function istwo-fold, in that the molecules are reactive with cell membrane antigenspresent on the surface of human carcinomas, and further in that theantibodies have the ability to internalize within the carcinoma cells,subsequent to binding, making them especially useful for formingantibody-drug and antibody-toxin conjugates. In their unmodified formthe antibodies also manifest cytotoxic properties at specificconcentrations.

U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumortherapy and prophylaxis. However, this antibody is an antinuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal antinuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal antinuclear autoantibody.

SUMMARY OF THE INVENTION

This application teaches a method for producing patient specificanti-cancer antibodies using a novel paradigm of screening. Theseantibodies can be made specifically for one tumor and thus make possiblethe customization of cancer therapy. Within the context of thisapplication, anti-cancer antibodies having either cell-killing(cytotoxic) or cell-growth inhibiting (cytostatic) properties willhereafter be referred to as cytotoxic. These antibodies can be used inaid of staging and diagnosis of a cancer, and can be used to treat tumormetastases.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existinganti-cancer antibodies. The patient will be conventionally staged butthe available antibodies can be of use in further staging the patient.The patient can be treated immediately with the existing antibodies, anda panel of antibodies specific to the tumor can be produced either usingthe methods outlined herein or through the use of phage displaylibraries in conjunction with the screening methods herein disclosed.All the antibodies generated will be added to the library of anti-cancerantibodies since there is a possibility that other tumors can bear someof the same epitopes as the one that is being treated.

In addition to anti-cancer antibodies, the patient can elect to receivethe currently recommended therapies as part of a multi-modal regimen oftreatment. The fact that the antibodies isolated via the presentmethodology are relatively non-toxic to non-cancerous cells allowcombinations of antibodies at high doses to be used, either alone, or inconjunction with conventional therapy. The high therapeutic index willalso permit re-treatment on a short time scale that should decrease thelikelihood of emergence of treatment resistant cells.

If the patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival andanti-cancer antibody conjugated red blood cells can be effective againstin situ tumors, too. Alternatively, the antibodies may be conjugated toother hematogenous cells, e.g. lymphocytes, macrophages, monocytes,natural killer cells, etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody dependent cellular cytotoxicity or complement dependentcytotoxicity. For example murine IgM and IgG2a antibodies can activatehuman complement by binding the C-1 component of the complement systemthereby activating the classical pathway of complement activation whichcan lead to tumor lysis. For human antibodies the most effectivecomplement activating antibodies are generally IgM and IgG1. Murineantibodies of the IgG2a and IgG3 isotype are effective at recruitingcytotoxic cells that have Fc receptors which will lead to cell killingby monocytes, macrophages, granulocytes and certain lymphocytes. Humanantibodies of both the IgG1 and IgG3 isotype mediate ADCC.

Another possible mechanism of antibody mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are two additional mechanisms of antibody mediated cancer cellkilling which are more widely accepted. The first is the use ofantibodies as a vaccine to induce the body to produce an immune responseagainst the putative cancer antigen that resides on the tumor cell. Thesecond is the use of antibodies to target growth receptors and interferewith their function or to down regulate that receptor so thateffectively its function is lost.

Accordingly, it is an objective of the invention to teach a method forproducing anti-cancer antibodies from cells derived from a particularindividual which are cytotoxic with respect to cancer cells whilesimultaneously being relatively non-toxic to non-cancerous cells.

It is an additional objective of the invention to produce novelanti-cancer antibodies.

It is a further objective of the instant invention to produceanti-cancer antibodies whose cytotoxicity is mediated through antibodydependent cellular toxicity.

It is yet an additional objective of the instant invention to produceanti-cancer antibodies whose cytotoxicity is mediated through complementdependent cellular toxicity.

It is still a further objective of the instant invention to produceanti-cancer antibodies whose cytotoxicity is a function of their abilityto catalyze hydrolysis of cellular chemical bonds.

Still an additional objective of the instant invention is to produceanti-cancer antibodies useful as a vaccine to produce an immune responseagainst putative cancer antigen residing on tumor cells.

A further objective of the instant invention is the use of antibodies totarget cell membrane proteins, such as growth receptors, cell membranepumps and cell anchoring proteins, thereby interfering with or downregulating their function.

Yet an additional objective of the instant invention is the productionof anti-cancer antibodies whose cell-killing utility is concomitant withtheir ability to effect a conformational change in cellular proteinssuch that a signal will be transduced to initiate cell-killing.

A still further objective of the instant invention is to produceanti-cancer antibodies which are useful for diagnosis, prognosis, andmonitoring of cancer, e.g. production of a panel of therapeuticanti-cancer antibodies to test patient samples to determine if theycontain any suitable antibodies for therapeutic use.

Yet another objective of the instant invention is to produce novelantigens, associated with cancer processes, which can be discovered byusing anti-cancer antibodies derived by the process of the instantinvention. These antigens are not limited to proteins, as is generallythe case with genomic data; they may also be derived from carbohydratesor lipids or combinations thereof.

Other objects and advantages of this invention will become apparent fromthe following description wherein are set forth, by way of illustrationand example, certain embodiments of this invention.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementherein described and shown. It will be apparent to those skilled in theart that various changes may be made without departing from the scope ofthe invention and the invention is not to be considered limited to whatis shown and described in the specification.

One of the potential benefits of monoclonal antibodies with respect tothe treatment of cancer is their ability to specifically recognizesingle antigens. It was thought that in some instances cancer cellspossess antigens that were specific to that kind of transformed cell. Itis now more frequently believed that cancer cells have few uniqueantigens, rather, they tend to over-express a normal antigen or expressfetal antigens. Nevertheless, the use of monoclonal antibodies provideda method of delivering reproducible doses of antibodies to the patientwith the expectation of better response rates than with polyclonalantibodies.

Traditionally, monoclonal antibodies have been made according tofundamental principles laid down by Kohler and Milstein. Mice areimmunized with antigens, with or without, adjuvants. The splenocytes areharvested from the spleen for fusion with immortalized hybridomapartners. These are seeded into microtitre plates where they can secreteantibodies into the supernatant that is used for cell culture. To selectfrom the hybridomas that have been plated for the ones that produceantibodies of interest the hybridoma supernatants are usually tested forantibody binding to antigens in an ELISA (enzyme linked immunosorbentassay) assay. The idea is that the wells that contain the hybridoma ofinterest will contain antibodies that will bind most avidly to the testantigen, usually the immunizing antigen. These wells are then subclonedin limiting dilution fashion to produce monoclonal hybridomas. Theselection for the clones of interest is repeated using an ELISA assay totest for antibody binding. Therefore, the principle that has beenpropagated is that in the production of monoclonal antibodies thehybridomas that produce the most avidly binding antibodies are the onesthat are selected from among all the hybridomas that were initiallyproduced. That is to say, the preferred antibody is the one with highestaffinity for the antigen of interest.

There have been many modifications of this procedure such as using wholecells for immunization. In this method, instead of using purifiedantigens, entire cells are used for immunization. Another modificationis the use of cellular ELISA for screening. In this method instead ofusing purified antigens as the target in the ELISA, fixed cells areused. In addition to ELISA tests, complement mediated cytotoxicityassays have also been used in the screening process. However,antibody-binding assays were used in conjunction with cytotoxicitytests. Thus, despite many modifications, the process of producingmonoclonal antibodies relies on antibody binding to the test antigen asan endpoint.

Most antibodies directed against cancer cells have been produced usingthe traditional methods outlined above. These antibodies have been usedboth therapeutically and diagnostically. In general, for both theseapplications, the antibody has been used as the targeting agent thatdelivers a payload to the site of the cancer. These antibody conjugatescan either be radioactive, toxic, or serve as an intermediary forfurther delivery of a drug to the body, such as an enzyme or biotin.Furthermore, it was widely held, until recently, that naked antibodieshad little effect in vivo. Both HERCEPTIN and RITUXIMAB are humanizedmurine monoclonal antibodies that have recently been approved for humanuse by the FDA. However, both these antibodies were initially made byassaying for antibody binding and their direct cytotoxicity was not theprimary goal during the production of hybridomas. Any tendency for theseantibodies to produce tumor cell killing is thus through chance, not bydesign.

Although the production of monoclonal antibodies have been carried outusing whole cell immunization for various applications the screening ofthese hybridomas have relied on either putative or identified targetantigens or on the selectivity of these hybridomas for specific tissues.It is axiomatic that the best antibodies are the ones with the highestbinding constants. This concept originated from the basic biochemicalprinciple that enzymes with the highest binding constants were the onesthat were the most effective for catalyzing a reaction. This concept isapplicable to receptor ligand binding where the drug molecule binding tothe receptor with the greatest affinity usually has the highestprobability for initiating or inhibiting a signal. However, this may notalways be the case since it is possible that in certain situations theremay be cases where the initiation or inhibition of a signal may bemediated through non-receptor binding. The information conveyed by aconformational change induced by ligand binding can have manyconsequences such as a signal transduction, endocytosis, among theothers. The ability to produce a conformational change in a receptormolecule may not necessarily be due to the filling of a ligand receptorpocket but may occur through the binding of another extra cellulardomain or due to receptor clustering induced by a multivalent ligand.

The production of antibodies to produce cell killing need not bepredicated upon screening of the hybridomas for the best bindingantibodies. Rather, although not advocated by those who producemonoclonal antibodies, the screening of the hybridoma supernatants forcell killing or alternatively for cessation of growth of the cancerouscells may be selected as a desirable endpoint for the production ofcytotoxic or cytostatic antibodies. It is well understood that thein-vivo antibodies mediate their function through the Fc portions andthat the utility of the therapeutic antibody is determined by thefunctionality of the constant region or attached moieties. In this casethe FAb portion of the antibody, the antigen-combining portion, willconfer to the antibody its specificity and the Fc portion itsfunctionality. The antigen combining site of the antibody can beconsidered to be the product of a natural combinatorial library. Theresult of the rearrangement of the variable region of the antibody canbe considered a molecular combinatorial library where the output is apeptide. Therefore, the sampling of this combinatorial library can bebased on any parameter. Like sampling a natural compound library forantibiotics, it is possible to sample an antibody library for cytotoxicor cytostatic compounds.

The various endpoints in a screen must be differentiated from eachother. For example, the difference between antibody binding to the cellis distinct from cell killing. Cell killing (cytotoxicity) is distinctfrom the mechanisms of cell death such as oncosis or apoptosis. Therecan be many processes by which cell death is achieved and some of thesecan lead either to oncosis or apoptosis. There is speculation that thereare other cell death mechanisms other than oncosis or apoptosis butregardless of how the cell arrives at death there are some commonalitiesof cell death. One of these is the absence of metabolism and another isthe denaturation of enzymes. In either case vital stains will fail tostain these cells. These endpoints of cell death have been longunderstood and predate the current understanding of the mechanisms ofcell death. Furthermore, there is the distinction between cytotoxiceffects where cells are killed and cytostatic effects where theproliferation of cells are inhibited.

In a preferred embodiment of the present invention, the assay isconducted by focusing on cytotoxic activity toward cancerous cells as anend point. In a preferred embodiment, a live /dead assay kit, forexample the LIVE/DEAD® Viability/Cytotoxicity Assay Kit (L-3224) byMolecular Probes, is utilized. The Molecular Probes kit provides atwo-color fluorescence cell viability assay that is based on thesimultaneous determination of live and dead cells with two probes thatmeasure two recognized parameters of cell viability intracellularesterase activity and plasma membrane integrity. The assay principlesare general and applicable to most eukaryotic cell types, includingadherent cells and certain tissues, but not to bacteria or yeast. Thisfluorescence-based method of assessing cell viability is preferred inplace of such assays as trypan blue exclusion, is Cr release and similarmethods for determining cell viability and cytotoxicity.

In carrying out the assay, live cells are distinguished by the presenceof ubiquitous intracellular esterase activity, determined by theenzymatic conversion of the virtually nonfluorescent cell-permeantCALCEIN AM to the intensely fluorescent Calcein. The polyanionic dyeCalcein is well retained within live cells, producing an intense uniformgreen fluorescence in live cells (ex/em ˜495 nm/˜515 nm). EthD-1 enterscells with damaged membranes and undergoes a 40-fold enhancement offluorescence upon binding to nucleic acids, thereby producing a brightred fluorescence in dead cells (ex/em ˜495 nm/˜635 nm). EthD-1 isexcluded by the intact plasma membrane of live cells. The determinationof cell viability depends on these physical and biochemical propertiesof cells. Cytotoxic events that do not affect these cell properties maynot be accurately assessed using this method. Background fluorescencelevels are inherently low with this assay technique because the dyes arevirtually nonfluorescent before interacting with cells.

In addition to the various endpoints for screening, there are two othermajor characteristics of the screening process. The library of antibodygene products is not a random library but is the product of a biasingprocedure. In the examples below, the biasing is produced by immunizingmice with fixed cells. This increases the proportion of antibodies thathave the potential to bind the target antigen. Although immunization isthought of as a way to produce higher affinity antibodies (affinitymaturation) in this case it is not. Rather, it can be considered as away to shift the set of antigen combining sites towards the targets.This is also distinct from the concept of isotype switching where thefunctionality, as dictated by the constant portion of the heavy chain,is altered from the initial IgM isotype to another isotype such as IgG.

The third key feature that is crucial in the screening process is theuse of multitarget screening. To a certain extent specificity is relatedto affinity. An example of this is the situation where an antigen hasvery limited tissue distribution and the affinity of the antibody is akey determinant of the specificity of the antibody—the higher theaffinity the more tissue specific the antibody and likewise an antibodywith low affinity may bind to tissues other than the one of interest.Therefore, to address the specificity issue the antibodies are screenedsimultaneously against a variety of cells. In the examples below thehybridoma supernatants (representing the earliest stages of monoclonalantibody development), are tested against a number of cell lines toestablish specificity as well as activity.

The antibodies are designed for therapeutic treatment of cancer inpatients. Ideally the antibodies can be naked antibodies. They can alsobe conjugated to toxins. They can be used to target other molecules tothe cancer. e.g. biotin conjugated enzymes. Radioactive compounds canalso be used for conjugation.

The antibodies can be fragmented and rearranged molecularly. For exampleFv fragments can be made; sFv-single chain Fv fragments; diabodies etc.

It is envisioned that these antibodies can be used for diagnosis,prognosis, and monitoring of cancer. For example the patients can haveblood samples drawn for shed tumor antigens which can be detected bythese antibodies in different formats such as ELISA assays, rapid testpanel formats etc. The antibodies can be used to stain tumor biopsiesfor the purposes of diagnosis. In addition a panel of therapeuticantibodies can be used to test patient samples to determine if there areany suitable antibodies for therapeutic use.

The hybridoma cell lines 1LN-8 (shown in table on page 30), 3BD-8 (shownin table on page 26), 3BD-26 (shown in table on page 26), 3BD-27 (shownin table on page 26), H460-27 (shown in table on page 45), H460-23(shown in table on page 45), 7BD-14 (shown in table on page 35) and5LAC20 (shown in table on page 41) where deposited, in accordance withthe Budapest Treaty, with the american Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va. 20110-2209 on Nov. 21, 2000 UnderAccession Numbers PTA-2693, PTA-2696, PTA-2695, PTA-2698, PTA-2699,PTA-2700, PTA-2697 and PTA-2694 respectively. The hybridoma cell linesH460-16-2 (shown in the table on page 45) and 7BDI-60 (shown in thetable on page 39) were deposited on Sep. 4, 2002 under Accession NumbersPTA-4621 and PTA-4623 respectively. In accordance with 37 CFR 1.808, thedepositors assure that all restrictions imposed on the availability tothe public of the deposited materials will be irrevocably removed uponthe granting of a patent. The depositors additionally assure that thedeposited materials will be replaced if viable samples cannot bedispensed by the depository.

EXAMPLE ONE

In order to produce monoclonal antibodies specific for a tumor samplethe method of selection of the appropriate hybridoma wells iscomplicated by the probability of selecting wells which will producefalse positive signals. That is to say that there is the likelihood ofproducing antibodies that can react against normal cells as well ascancer cells. To obviate this possibility one strategy is to mask theanti-normal antigen antibodies from the selection process. This can beaccomplished by removing the anti-normal antibodies at the first stageof screening thereby revealing the presence of the desired antibodies.Subsequent limiting dilution cloning can delineate the clones that willnot produce killing of control cells but will produce target cancer cellkilling.

Biopsy specimens of breast, melanoma, and lung tumors were obtained andstored at −70° C. until used. Single cell suspensions were prepared andfixed with −30° C., 70% ethanol, washed with PBS and reconstituted to anappropriate volume for injection. Balb/c mice were immunized with2.5×10⁵−1×10⁶ cells and boosted every third week until a finalpre-fusion boost was performed three days prior to the splenectomy. Thehybridomas were prepared by fusing the isolated splenocytes with Sp2/0and NS1 myeloma partners. The supernatants from the fusions were testedfor subcloning of the hybridomas. Cells (including A2058 melanoma cells,CCD-12CoN fibroblasts, MCF-12A breast cells among others) were obtainedfrom ATCC and cultured according to enclosed instructions. The HEY cellline was a gift from Dr. Inka Brockhausen. The non-cancer cells, e.g.CCD-12CoN fibroblasts and MCF-12A breast cells, were plated into 96-wellmicrotitre plates (NUNC) 1 to 2 weeks prior to screening. The cancercells, e.g. HEY, A2058, BT 483, and HS294t, were plated two or threedays prior to screening.

The plated normal cells were fixed prior to use. The plates were washedwith 100 microliters of PBS for 10 minutes at room temperature and thenaspirated dry. 75 microliters of 0.01 percent glutaraldehyde diluted inPBS were added to each well for five minutes and then aspirated. Theplates were washed with 100 microliters of PBS three times at roomtemperature. The wells were emptied and 100 microliters of one percenthuman serum albumin in PBS was added to each well for one hour at roomtemperature. The plates were then stored at four degrees Celsius.

Prior to the transfer of the supernatant from the hybridoma plates thefixed normal cells were washed three times with 100 microliters of PBSat room temperature. After aspiration to the microliters of the primaryhybridoma culture supernatants were transferred to the fixed cell platesand incubated for two hours at 37 degrees Celsius in a 8 percent CO₂incubator. The hybridoma supernatants derived from melanoma wasincubated with CCD-12 CoN cells and those derived from breast cancerwere incubated with MCF-12a cells.

After incubation the absorbed supernatant was divided into two 75microliter portions and transferred to target cancer cell plates. Priorto the transfer the cancer cell plates were washed three times with 100microliters of PBS. The supernatant from the CCD-12 CoN cells weretransferred to the A2058 and the HS294t cells, whereas the supernatantfrom MCF-12A cells were transferred to the HEY and BT 483 cells. Thecancer cells were incubated with the hybridoma supernatants for 18 hoursat 37 degrees Celsisu in an 8 percent CO₂ incubator.

The Live/Dead cytotoxicity assay was obtained from Molecular Probes(Eu,Oreg.). The assays were performed according to the manufacturer'sinstructions with the changes outlined below. The plates with the cellswere washed once with 100 microliters of PBS at 37° C. 75 to 100microliters of supernatant from the hybridoma microtitre plates weretransferred to the cell plates and incubated in a 8% CO₂ incubator for18-24 hours. Then, the wells that served as the all dead control wereaspirated until empty and 50 microliters of 70% ethanol was added. Theplate was then emptied by inverting and blotted dry. Room temperaturePBS was dispensed into each well from a multichannel squeeze bottle,tapped three times, emptied by inversion and then blotted dry. 50microliters of the fluorescent Live/Dead dye diluted in PBS was added toeach well and incubated at 37° C. in a 5% CO₂ incubator for one hour.The plates were read in a Perkin-Elmer HTS7000 fluorescence plate readerand the data was analyzed in Microsoft Excel.

Four rounds of screening were conducted to produce single clonehybridoma cultures. For two rounds of screening the hybridomasupernatants were tested only against the cancer cells. In the lastround of screening the supernatant was tested against a number ofnon-cancer cells as well as the target cells indicated in table 1. Theantibodies were isotyped using a commercial isotyping kit.

A number of monoclonal antibodies were produced in accordance with themethod of the present invention. These antibodies, whose characteristicsare summarized in Table 1, are identified as 3BD-3, 3BD-6, 3BD-8, 3BD-9,3BD-15, 3BD-25, 3BD-26 and 3BD-27. Each of the designated antibodies isproduced by a hybridoma cell line deposited with the American TypeCulture Collection at 10801 University Boulevard, Manassas, Va. havingan ATCC Accession Number as follows:

Antibody ATCC Accession Number 3BD-3 not deposited 3BD-6 not deposited3BD-8 PTA-2696 3BD-9 not deposited 3BD-15 not deposited 3BD-25 notdeposited 3BD-26 PTA-2695 3BD-27 PTA-2698

These antibodies are considered monoclonal after four rounds of limitingdilution cloning. The anti-melanoma antibodies did not producesignificant cancer cell killing. The panel of anti-breast cancerantibodies killed 32-87% of the target cells and <1-3% of the controlcells. The predominant isotype was IgG1 even though it was expected thatthe majority of anti-tumor antibodies would be directed againstcarbohydrate antigens, and thus, be of the IgM type. There is a hightherapeutic index since most antibodies spare the control cells fromcell death.

TABLE 1 Anti-Breast Cancer Antibodies % Cell Death Target forAnti-Breast Normal Fibroblast Cancer Antibodies Cells Fibrocystic BreastCells Clones (HEY & A2058) (CCD-12CoN) (NCF-12A) Isotype 3BD-3 74.9%3.7%  <1% y1, λ 3BD-6 68.5% 5.6%  <1% y1, λ 3BD-8 81.9% 4.5% 2.6% y1, κ3BD-9 77.2% 7.9%  <1% y1, λ 3BD-15 87.1%  <1%  <1% y1, λ 3BD-26 54.8%3.3%  <1% μ, κ 2BD-25 32.4% 3.6%  <1% y1, κ 3BD-27 60.1% 8.3% 1.3% y1, κ

EXAMPLE 2

In this example customized anti-cancer antibodies are produced by firstobtaining samples of the patient's tumor. Usually this is from a biopsyspecimen from a solid tumor or a blood sample from hematogenous tumors.The samples are prepared into single cell suspensions and fixed forinjection into mice. After the completion of the immunization schedulethe hybridomas are produced from the splenocytes. The hybridomas arescreened against a variety of cancer cell lines and normal cells instandard cytotoxicity assays. Those hybridomas that are reactive againstcancer cell lines but are not reactive against normal non-transformedcells are selected for further propagation. Clones that were consideredpositive were ones that selectively killed the cancer cells but did notkill the non-transformed cells. The antibodies are characterized for alarge number of biochemical parameters and then humanized fortherapeutic use. The melanoma tumor cells isolated and cell lines werecultured as described in Example 1. Balb/c mice were immunized accordingto the following schedule: 200,000 cells s.c. and i.p. on day 0, then200,000 cells were injected i.p. on day 21, then 1,000,000 cells wereinjected on day 49, then 1,250,000 cells in Freund's Complete Adjuvantwere injected i.p. on day 107, and then 200,000 cells were injected onday 120 i.p. and then the mice were sacrificed on day 123. The spleenswere harvested and the splenocytes were divided into two aliquots forfusion with Sp2/0 (1LN) or NS-1 (2LN) myeloma partners using the methodsoutlined in example 1.

The screening was carried out 11 days after the fusion against A2058melanoma cells and CCD-12CoN fibroblasts. Each pair of plates werewashed with 100 microliters of room temperature PBS and then aspiratedto near dryness. Then 50 microliters of hybridoma supernatant was addedto the same wells on each of the two plates. The spent Sp2/0 supernatantwas added to the control wells at the same volume and the plates wereincubated for around 18 hours at 37 degrees Celsius at a 8% CO₂, 98%relative humidity incubator. Then each pair of plates were removed andin the positive control wells 50 microliters of 70% ethanol wassubstituted for the media for 4 seconds. The plates were then invertedand washed with room temperature PBS once and dried. Then 50 uL offluorescent live/dead dye diluted in PBS (Molecular Probes Live/DeadKit) was added for one hour and incubated at 37 degrees Celsius. Theplates were then read in a Perkin-Elmer fluorescent plate reader and thedata analyzed using Microsoft Excel. The wells that were consideredpositive were subcloned and the same screening process was repeated 13days later and then 33 days later. The results of the last screening isoutlined in Table 2 below. A number of monoclonal antibodies wereproduced in accordance with the method of the present invention. Theseantibodies, whose characteristics are summarized in Table 2, areidentified as 1LN-1, 1LN-8, 1LN-12, 1LN-14, 2LN-21, 2LN-28, 2LN-29,2LN-31, 2LN-33, 2LN-34 and 2LN-35. Each of the designated antibodies isproduced by a hybridoma cell line deposited with the American TypeCulture Collection at 10801 University Boulevard, Manassas, Va. havingan ATCC Accession Number as follows:

Antibody ATCC Accession Number 1LN-1 not deposited 1LN-8 PTA-2693 1LN-12not deposited 1LN-14 not deposited 2LN-21 not deposited 2LN-28 notdeposited 2LN-29 not deposited 2LN-31 not deposited 2LN-33 not deposited2LN-34 not deposited 2LN-35 not deposited

TABLE 2 Anti-Melanoma Antibodies % Cell Death Target for Anti- MelanomaNormal Fibroblast Antibodies Cells Clones (A2058) (CCD-1 2CoN) 1LN-1 59.4%   <1% 1LN-8  11.0%  5.0% 1LN-12  55.2%  1.4% 1LN-14  51.4%   <1%2LN-21  72.0% 15.9% 2LN-28  66.6% 12.4% 2LN-29  78.2%  6.1% 2LN-31 100% 7.8% 2LN-33  94.2%   <1% 2LN-34  56.6% 11.2% 2LN-35  66.5%  5.6%

The table illustrates that clones from both the Sp2/0 and NS-1 fusionswere able to produce antibodies that had a greater than 50% killing ratefor cancerous cells and at the same time some of the clones were able toproduce less than one percent killing of normal control fibroblasts.

EXAMPLE 3

In this example antibodies were produced to several different breasttumor samples following the method of Example 2 in order to demonstratethe generality of producing customized antibodies. Biopsy specimens ofbreast tumors were obtained and stored at −70° C. until used as noted inExample 1. Single cell suspensions were prepared for each specimen andfixed with −30° C., 70% ethanol, washed with PBS and reconstituted to anappropriate volume for injection. Female, 7-8 week old, A strain,H-2^(d) haplotype Balb/c mice (Charles River Canada, St. Constant, QC,Can), were immunized with 2.5×10⁵-1×10⁶ cells and boosted every thirdweek until a final pre-fusion boost was performed three days prior tothe splenectomy. The hybridomas were prepared by fusing the isolatedsplenocytes with Sp2/0 myeloma partners. The supernatants from thefusions were tested for subcloning of the hybridomas.

Hs574.T breast ductal carcinoma cells, A2058 melanoma cells, NCI-H460human lung large cell carcinoma, NCI-H661 human lung large cellcarcinoma, CCD-112CoN human colon fibroblasts, CCD-27sk human skinfibroblasts, MCF-12A human mammary epithelial cells, Hs574.mg humanbreast cells and other cell lines, were obtained from ATCC and culturedaccording to enclosed instructions. Both cancer and non-cancer cellswere plated three to four days prior to screening.

The hybridomas were cultured for ten to twelve days after fusion andobserved under the microscope. When 20 to 25% of the wells were over 80%confluent, the hybridoma supernatants were screened in a cytotoxicityassay. The hybridoma supernatants were divided into two 75 microliterportions; one portion was added to a target cancer cell plate andanother to a non-cancer cell plate. Prior to transfer of hybridomasupernatants, the cell plates were washed three times with 100microliters of PBS. The supernatant from the anti-breast cancerhybridomas were transferred to the Hs574.T and the Hs574.mg cells,whereas the supernatant from the anti-lung cancer hybridoma weretransferred to the NCI-H460 and CCD-27SK cells. The cancer cells wereincubated with the hybridoma supernatants for 18 hours at 37 degreesCelsius in an 8 percent CO₂ incubator.

The Live/Dead cytotoxicity assay was obtained from Molecular Probes(Eugene, Oreg.). The assays were performed according to themanufacturer's instructions with the changes outlined below. The plateswith the cells were washed once with 100 microliters of PBS at 37° C. 75to 100 microliters of supernatant from the hybridoma microtitre plateswere transferred to the cell plates and incubated in a 8% CO₂ incubatorfor 18-24 hours. Then, the wells that served as the dead control cellswere aspirated until empty and 50 microliters of 70% ethanol was added.The plate was then emptied by inverting and blotted dry. Roomtemperature PBS was dispensed into each well from a multichannel squeezebottle, tapped three times, emptied by inversion and then blotted dry.50 microliters of the fluorescent Live/Dead dye diluted in PBS was addedto each well and incubated at 37° C. in a 5% CO₂ incubator for one hour.The plates were read in a Perkin-Elmer HTS7000 fluorescence plate readerand the data was analyzed in Microsoft Excel (Microsoft, Redmond,Wash.).

Four rounds of screening were conducted to produce single clonehybridoma cultures. For two rounds of screening the hybridomasupernatants were tested only against the cancer cells. In the lastround of screening the supernatant was tested against a number ofnon-cancer cells as well as the target cells indicated in Table 3. Theantibodies were isotyped using a commercial isotyping kit (Roche,Indianapolis, Ind.).

A number of monoclonal antibodies were produced in accordance with themethod of the present invention. These antibodies, whose characteristicsare summarized in Table 3, are identified as 4BD-1, 4BD-3, 4BD-6, 4BD-9,4BD-13, 4BD-18, 4BD-20, 4BD-25, 4BD-37, 4BD-32, 4BD-26, 4BD-27, 4BD-28,4BD-50, 6BD-1, 6BD-3, 6BD-5, 6BD-11, 6BD-25, 7BD-7, 7BD-12-1, 7BD-12-2,7BD-13, 7BD-14, 7BD-19, 7BD-21, 7BD-24, 7BD-29, 7BD-30, 7BD-31, 7BDI-17,7BDI-58, 7BDI-60 and 7BDI-62. Each of the designated antibodies isproduced by a hybridoma cell line deposited with the American TypeCulture Collection at 10801 University Boulevard, Manassas, Va. havingan ATCC Accession Number as follows:

Antibody ATCC Accession Number 4BD-1 not deposited 4BD-3 not deposited4BD-6 not deposited 4BD-9 not deposited 4BD-13 not deposited 4BD-18 notdeposited 4BD-20 not deposited 4BD-25 not deposited 4BD-37 not deposited4BD-32 not deposited 4BD-26 not deposited 4BD-27 not deposited 4BD-28not deposited 4BD-50 not deposited 6BD-1 not deposited 6BD-3 notdeposited 6BD-5 not deposited 6BD-11 not deposited 6BD-25 not deposited7BD-7 not deposited 7BD-12-1 not deposited 7BD-12-2 not deposited 7BD-13not deposited 7BD-14 PTA-2697 7BD-19 not deposited 7BD-21 not deposited7BD-24 not deposited 7BD-29 not deposited 7BD-30 not deposited 7BD-31not deposited 7BDI-17 not deposited 7BDI-58 not deposited 7BDI-60PTA-4623 7BDI-62 not deposited

These antibodies are considered monoclonal after four rounds of limitingdilution cloning. The panel of anti-breast cancer antibodies killed15-79% of the target cells and <1-31% of the control cells. The majorityof anti-tumor antibodies were IgM type, suggesting they could bedirected against carbohydrate antigens on the surface of tumor cells.There is a high therapeutic index since most of the antibodies do notcause the normal cells to undergo cell death.

These monoclonal antibodies are characterized for a number ofimmunological and biochemical parameters. A cell based enzyme linkedimmunosorbent assay (ELISA) was established for measuring the binding ofthe antibodies derived of each clones to different cell lines. Cellswere seeded and grown on 96-well tissue culture plates. The plates werewashed with 100 microliters of PBS. 100 microliters of cold 4 percentparaformaldehyde in PBS were added to each well for ten minutes and thenaspirated. The plates were washed with PBS using a multichannel squeezebottle. The wells were emptied and 100 microliters of blocking buffer (1percent hydrocasein, 0.1 percent geletin in 50 mM Tris-HCl buffer, pH9.3) was added to each well for one hour at room temperature. The plateswere washed three times with a buffer (0.05 percent Tween 20 in 10 mMPBS) at room temperature and then stored at −30 degrees Celsius with 100microliters of the buffer. Prior to use the plates were thawed and thebuffer was aspirated from each well. 75 microliters of hybridomasupernatant were added to each well and incubated for 60 minutes at roomtemperature. After the plates were washed with PBS using a multichannelsqueeze bottle, 50 microliters of a combination of peroxidase conjugatedgoat anti-mouse IgG and peroxidase conjugated donkey anti-mouse IgM(Jackson ImmunoResearch Lab, Inc., West Grove, Pa.) was added andincubated for 30 minutes at room temperature. After the last wash, 50microliters of orthophenylene diamine (OPD) (Sigma, St. Louis, Mo.) wasadded to each well and the optical density was read at 492 nm on theHTS7000 plate reader after adding equal volume of 1 N sulfuric acid.Different clones show different profiles in binding to different cells(Table 3). This indicates that they may target different cell surfaceantigen and further suggests the variable distribution of these antigenon the surface of cancer cells. Those binding only to cancer cells butnot to normal cells could identify certain tumor-associated antigen.

Table 3. Anti-Breast Cancer Antibodies

TABLE 3 Anti-Breast Cancer Antibodies % Cell Death Binding to cell linesClones Isotype Hs574.T Hs574.mg Hs574.T Hs574.mg NCl-H460 CCD-27sk A20586BD-1 μ, κ 38.2 5 0.8 0.5 0.6 0.3  ND* 6BD-3 μ, κ 79 12 0.35 0.25 0.240.14 ND 6BD-5 μ, κ 57.3 8 1.0 0.3 0.14 0.25 ND 6BD-11 μ, κ 52.3 11 0.150.1 0.17 0.1 ND 6BD-25 μ, κ 33.3 2 0.15 0.1 0.2 0.1 ND 4BD-26 μ, κ 271.8 0.5 ND ND <0.1 ND 4BD-27 μ, κ 19.6 <1 0.9 ND ND 0.5 ND 4BD-28 μ, κ26.4 <1 0.8 ND ND <0.1 ND 4BD-32 μ, κ 41.7 4 0.8 ND ND <0.1 ND 4BD-50 μ,κ 20 <1 0.8 ND ND <0.1 ND 4BD-1 μ, κ 23 31 0.6 ND ND <0.1 ND 4BD-3 μ, κ29.7 8.2 1.7 ND ND 0.1 ND 4BD-6 μ, κ 17 <1 0.9 ND ND <0.1 ND 4BD-9 μ, κ15 <1 0.6 ND ND <0.1 ND 4BD-13 μ, κ 31 <1 1.2 ND ND <0.1 ND 4BD-18 μ, κ23.3 2.4 0.7 ND ND 0.12 ND 4BD-20 μ, κ 45 <1 0.95 ND ND <0.1 ND 4BD-25μ, κ 26 14.16 1.8 ND ND 0.1 ND 4BD-37 μ, κ 30 <1 0.8 ND ND <0.1 ND 7BD-7μ, κ 24 3 0.8 0.3 1.4 0.26 ND 7BD-12-1 μ, κ 22 6 0.36 0.16 0.43 0.1 ND7BD-12-2 μ, κ 31 2 0.2 0.2 0.2 0.2 0.2 7BD-13 μ, κ 29 12 0.1 0.15 0.20.1 0.2 7BD-14 μ, κ 32 13 0.4 0.4 0.6 0.3 0.5 7BD-19 μ, κ 20 4 1.3 0.40.43 0.2 ND 7BD-21 μ, κ 21 13 0.4 0.5 0.25 0.3 ND 7BD-24 μ, κ 32 15 0.30.1 0.14 0.15 ND 7BD-29 15 16 0.3 0.24 0.14 0.16 ND 7BD-30 μ, κ 23 130.34 0.24 0.2 0.16 ND 7BD-31 μ, κ 28 10 0.3 0.4 0.4 0.3 0.4 7BDI-17 μ, κ23 <1 0.75 ND ND ND ND 7BDI-58 γ1, κ 17.5 <1 0.77 ND ND ND ND 7BDI-60γ1, κ 15 <1 0.73 ND ND ND ND 7BDI-62 15 5 0.55 ND ND ND ND *ND: notdone.

EXAMPLE 4

In this example customized anti-cancer antibodies are produced to a lungcancer sample by first obtaining samples of the patient's tumorpreparing single cell suspensions which are then fixed for injectioninto mice as noted in Example 1. After the completion of theimmunization schedule the hybridomas are produced from the splenocytes.The hybridomas are screened against a variety of cancer cell lines andnormal cells in standard cytotoxicity assays. Those hybridomas that arereactive against cancer cell lines but are not reactive against normalnon-transformed cells are selected for further propagation. Clones thatwere considered positive were ones that selectively killed the cancercells but did not kill the non-transformed cells.

The lung cancer cells were isolated and cell lines were cultured asdescribed in Example 1. Female, 7-8 week old, A strain, H-2^(d)haplotype Balb/c mice (Charles River Canada, St. Constant, QC, Can),were immunized with human lung cancer cells. The lung cancer cellsuspensions were emulsified in an equal volume of Freund's completeadjuvant (FCA) for the first immunization and then in Freund'sincomplete adjuvant (FIA) for subsequent immunizations at 0, 21, 45days. 5×10⁵ cells were used to immunize each mouse either through asubcutaneous or intra-peritoneal route. Immunized mice were sacrificed3-4 days after the final immunization with human lung cancer cells at148 days, given intra-peritoneally, in PBS at pH 7.4. The spleens wereharvested and the splenocytes were divided into two aliquots for fusionwith Sp2/0 myeloma partners using the methods outlined in Example 1.

The screening was carried out 10 days after the fusion against NCI-H460and/or NCI-H661 cells and CCD-27SK fibroblasts. Each pair of plates werewashed with 100 microliters of room temperature PBS and then aspiratedto near dryness. Then 75 microliters of hybridoma supernatant was addedper well on each of the two plates. The spent Sp2/0 supernatant wasadded to the control wells at the same volume and the plates wereincubated for around 18 hours at 37 degrees Celsius at a 8%CO₂, 98%relative humidity incubator. Then each pair of plates was removed and inthe positive control wells 50 microliters of 70% ethanol was substitutedfor the media for 4 seconds. The plates were then inverted and washedwith room temperature PBS once and dried. Then 50 microliters offluorescent live/dead dye diluted in PBS (Molecular Probes Live/DeadKit) was added for one hour and incubated at 37 degrees Celsius. Theplates were then read in a Perkin-Elmer fluorescent plate reader and thedata analyzed using Microsoft Excel. The wells that were consideredpositive were subcloned and the same screening process was repeated 6days later and then 13 days later. The result of the last screening isoutlined in Table 4 below. Antibodies were characterized for binding todifferent cell lines with a cellular ELISA according to the methods ofExample 3. A number of monoclonal antibodies were produced in accordancewith the method of the present invention. These antibodies, whosecharacteristics are summarized in Table 4, are identified as 5LAC2,5LAC4, 5LAC20, and 5LAC23. Each of the designated antibodies is producedby a hybridoma cell line deposited with the American Type CultureCollection at 10801 University Boulevard, Manassas, Va. having an ATCCAccession Number as follows:

Antibody ATCC Accession Number 5LAC2 not deposited 5LAC4 not deposited5LAC20 PTA-2694 5LAC23 not deposited

TABLE 4 Anti-Lung Cancer Antibodies % Cell Death Binding to cell linesClones Isotype Hs574.T NCl-H460 NCl-H661 A2058 CCD-27sk Hs574.T Hs574.mgNCl-H460 CCD-27sk A2058 5LAC2 μ, κ 30  7 45.3 23 <1 0.2 0.2 0.26 0.2 0.25LAC4 μ, κ 21 11 20.5 23 3 0.7 0.9 1.7 0.8 0.9 5LAC20 μ, κ 23  7 66.4 243 0.5 0.2 0.6 0.2 0.2 23  8 57.6 25 5 0.6 0.6 0.6 0.6 0.6 *ND: not done

The table illustrates that clones were able to produce antibodies thathad a greater than 7-67% killing rate for cancerous cells and at thesame time some of the clones were able to produce less than five percentkilling of normal control fibroblasts.

EXAMPLE 5

In this example customized anti-cancer antibodies are produced to apatient's lung cancer cells, but cultured cells were used in theantibody development process to demonstrate the generality of theimmunization process. The samples were prepared into single cellsuspensions and fixed for injection into mice as noted in Example 1.After the completion of three rounds of immunization with cells deriveddirectly from a patient's lung cancer, the mice were immunized twicewith a human lung large cell carcinoma cell line (NCI-H460). Hybridomaswere produced from splenocytes and the supernatants were screenedagainst a variety of cancer cell lines and normal cells in standardcytotoxicity assays. Those hybridomas that were reactive against cancercell lines but were not reactive against normal non-transformed cellswere selected for further propagation. Clones that were consideredpositive were ones that selectively killed the cancer cells but did notkill the non-transformed cells. The antibodies are characterized for alarge number of biochemical parameters and then humanized fortherapeutic use.

The lung tumor cells isolated and cell lines were cultured as describedin Example 1. Balb/c mice, A strain with H-2^(d) haplotype from CharlesRiver Canada, St. Constant, Quebec, Canada, female, 7-8 week old, wereimmunized with the human lung cancer cells emulsified in an equal volumeof either Freund's complete adjuvant (FCA) for the first immunizationand then in Freund's incomplete adjuvant (FIA) for subsequentimmunizations at 0, 21, 45 days with 5×10⁵ cells. The mice wereimmunized with fixed NCI H460 cells, which were prepared from NCI H460cells grown in T-75 cell culture flask by scraping mono-layer cells intocell suspensions at 105, 150 and 170 days. Immunized mice weresacrificed 3-4 days after the final immunization with NCI H460 cells,given intra-peritoneally, in phosphate buffered saline buffer (PBS), pH7.4. The spleens were harvested and the splenocytes were divided intotwo aliquots for fusion with Sp2/0 myeloma partners using the methodsoutlined in Example 1.

The screening was carried out 10 days after the fusion against NCI H460cells and CCD-27SK fibroblasts as described in Example 4. Antibodieswere characterized for binding to different cell lines with a cellularELISA according to the methods of Example 3.

The wells that were considered positive were subcloned and the samescreening process was repeated 9 days and 18 days later. The results areoutlined in Table 5 below. A number of monoclonal antibodies wereproduced in accordance with the method of the present invention. Theseantibodies, whose characteristics are summarized in Table 5, areidentified as H460-1, H460-4, H460-5, H460-10, H460-14, H460-16-1,H460-16-2, H460-23 and H460-27. Each of the designated antibodies isproduced by a hybridoma cell line deposited with the American TypeCulture Collection at 10801 University Boulevard, Manassas, Va. havingan ATCC Accession Number as follows:

Antibody ATCC Accession Number H460-1 not deposited H460-4 not depositedH460-5 not deposited H460-10 not deposited H460-14 not depositedH460-16-1 not deposited H460-16-2 PTA-4621 H460-23 PTA-2700 H460-27PTA-2699

TABLE 5 Anti-Lung Cancer Antibodies % Cell Death Binding to cell linesClones Isotype NCl-H460 Hs574.T A2058 CCD- Hs574. Hs574.m NCl- CCD-A2058 H460-1 γ1, ê 16 30 23 <1 1.0 0.6 0.5 0.7 ND H460-4 γ1, ê 37 21 233 1.0 0.6 0.4 0.6 ND H460-5 μ, κ 22.5 23 24 3 1.0 0.3 0.3 0.2 ND H460-10μ, κ 8 23 25 5 0.3  0.14 0.2 0.1 ND H460-14 γ1, ê 17 ND ND 4 1.1 0.6 0.40.54 ND H460-16-1 γ1, ê 33 ND ND 8 1.0 0.6 0.3 0.5 ND H460-16-2 γ1, ê 22ND ND 3 1.0 0.6 0.3 0.7 ND H460-22-1 γ1, ê 21 ND ND 5 0.6 0.4 0.3 0.4 NDH460-22-2 μ, κ 23 ND ND 3 0.4 0.1 0.1 0.1 ND H460-23 μ, κ 36 36 18 1 0.41.1 0.54 0.53 0.58 H460-27 μ, κ 33 31 16 8 0.3 0.4 0.4 0.3 0.4  *ND: notdone

The table illustrates that clones were able to produce antibodies thathad a greater than 15% killing rate for cancerous cells and at the sametime some of the clones were able to produce less than eight percentkilling of normal control fibroblasts.

The anti-cancer antibodies of the invention are useful for treating apatient with a cancerous disease when administered in admixture with apharmaceutically acceptable adjuvant, for example normal saline, a lipidemulsion, albumen, phosphate buffered saline or the like and areadministered in an amount effective to mediate treatment of saidcancerous disease, for example with a range of about 1 microgram permilliliter to about 1 gram per milliliter.

The method for treating a patient suffering from a cancerous disease mayfurther include the use of conjugated anti-cancer antibodies and wouldthis include conjugating patient specific anti-cancer antibodies with amember selected from the group consisting of toxins, enzymes,radioactive compounds, and hematogenous cells; and administering theseconjugated antibodies to the patient; wherein said anti-cancerantibodies are administered in admixture with a pharmaceuticallyacceptable adjuvant, for example normal saline, a lipid emulsion,albumen, phosphate buffered saline or the like and are administered inan amount effective to mediate treatment of said cancerous disease, forexample with a range of about 1 microgram per mil to about 1 gram permil. In a particular embodiment, the anti-cancer antibodies useful ineither of the above outlined methods may be a humanized antibody.

The anti-cancer antibodies of the invention are useful for treating apatient with a cancerous disease when administered in admixture with apharmaceutically acceptable adjuvant, for example normal saline, a lipidemulsion, albumen, phosphate buffered saline or the like and areadministered in an amount effective to mediate treatment of saidcancerous disease, for example with a range of about 1 microgram per milto about 1 gram per mil.

The method for treating a patient suffering from a cancerous disease mayfurther include the use of conjugated anti-cancer antibodies and wouldthis include conjugating patient specific anti-cancer antibodies with amember selected from the group consisting of toxins, enzymes,radioactive compounds, and hematogenous cells; and administering theseconjugated antibodies to the patient; wherein said anti-cancerantibodies are administered in admixture with a pharmaceuticallyacceptable adjuvant, for example normal saline, a lipid emulsion,albumen, phosphate buffered saline or the like and are administered inan amount effective to mediate treatment of said cancerous disease, forexample with a range of about 1 microgram per mil to about 1 gram permil. In a particular embodiment, the anti-cancer antibodies useful ineither of the above outlined methods may be a humanized antibody.

What is claimed is:
 1. An isolated monoclonal antibody or antigenbinding fragments thereof encoded by the clone deposited with the ATCCas Accession Number PTA-2700.
 2. The isolated clone deposited with theATCC as Accession Number PTA-2700.